The serine/threonine kinase glycogen synthase kinase-3 beta (GSK-3b) is a known master regulator for multiple cellular pathways that include insulin signaling and glycogen synthesis, neurotrophic factor signaling, and Wnt signaling. Consequently, this enzyme has a critical role in metabolism, transcription, development, and neuronal functions and has been implicated in multiple human disorders including Alzheimer's disease, bipolar disorder, noninsulin-dependent diabetes mellitus, cardiac hypertrophy, and cancer. Precisely how GSK-3b maintains its pathway specificity efficiently at the crossroads of many cellular processes is unclear. This regulation may involve allosteric sites within distinct structural domains of this complex kinase. The majority of the existing chemical inhibitors compete for the ATP-binding site of GSK-3b and inhibit additional kinases, while the two known structural series of ATP-noncompetitive inhibitors have low potency and suboptimal pharmacological properties that limit their use. Small molecules targeting the allosteric sites of GSK-3b could have the potential to provide highly specific GSK-3b inhibitors that may help elucidate GSK-3b function and regulation in distinct cellular pathways. Hence we propose a primary screen and a cascade of secondary biochemical and cellular assays to identify ATP-non competitive, allosteric inhibitors of GSK-3b. The anticipated high specificity of such probes could lead to the selective modulation of distinct cellular pathways dependent on GSK-3b. This probe development plan includes: 1) A primary kinase activity assay that will identify ATP-competitive as well as ATP- noncompetitive inhibitors that affect GSK-3b kinase activity. 2) A secondary kinase activity assay based on time resolved fluorescence (HTRF) detection of ADP that will distinguish allosteric modulators and ATP-competitive inhibitors. 3) Binding-based assays that will more precisely define a compound's mode of action. 4) Importantly, we have established a number of cellular assays that will help differentiate allosteric inhibitors and identify pathway-specific GSK-3b inhibitors. Ultimately, we plan to optimize the pharmacokinetic properties of pathway-specific, allosteric GSK-3b inhibitors, and test them in the established rodent models of mood and memory. PUBLIC HEALTH RELEVANCE: Glycogen synthase kinase-3 beta is a master regulator of multiple cell signaling pathways and is implicated in multiple human disorders. We propose to screen the MLPCN library to identify potent and selective allosteric modulators of GSK-3b. Collaborating with MLPCN and Stanley Center chemists, we will optimize the hit compounds to generate chemical probes to elucidate GSK-3b biology in distinct cellular pathways.